A major problem in the commercial cultivation of algae and cyanobacteria in axenic culture in bioreactors or open or closed ponds is that they can become contaminated by other, highly competitive but unwanted species of algae and cyanobacteria, fungi and bacteria, as well as by rotifers and other zooplankton that devour the desired species in the cultures. (Sheehan et al. 2004). Fluridone is the only USEPA-approved systemic herbicide that is commonly used for control of aquatic weeds (but not algae) in large bodies of water. It is a noncompetitive inhibitor of the enzyme phytoene desaturase (PDS), which is one of the first dedicated enzymes of the plant carotenoid biosynthesis pathway. Under high light intensities, carotenoids stabilize the photosynthetic apparatus by quenching the excess excitation energy; therefore, inhibition of phytoene desaturase decreases colored carotenoid concentration and causes photo-bleaching of green tissues (Böger and Sandmann 1998).
The pds gene was cloned from the herbicide-susceptible as well as from the recently-evolved, herbicide-resistant biotypes of the water weed hydrilla [Hydrilla verticillata (Lf) Royle]. Three separate and independent single-point mutations of the codon 304 encoding for Arg (Arg304) in pds were identified in the resistance biotypes (Michel et. al., 2004; Michel et. al., 2004 Patent application WO/2004/007691). The codon usage for Arg304 in the wild-type Hydrilla is CGT and single-point mutations yielding either Ser (AGT), Cys (TGT), or His (CAT) substitutions were identified in the fluridone resistance biotypes of Hydrilla. The resistant biotypes had biomass and-β carotene accumulations of up to 72% and 77% of the content in untreated plants, respectively, while in the susceptible population, fluridone strongly inhibited biomass accumulation and β-carotene accumulation, showing only 10% of the levels found in untreated plants (Michel, et al. 2004). Many fungi and bacteria that synthesize carotenoids as a photoprotectant are sensitive to PDS inhibitors.
Protoporphyrinogen oxidase (PPO; protox) is the last common enzyme in the tetrapyrrole biosynthetic pathway that produces heme and chlorophyll (Beale & Weinstein, 1990). In plants chlorophyll biosynthesis takes place exclusively in plastids, whereas heme is produced in both plastids and mitochondria. In both organelles, PPO converts protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX). Two different nuclear genes, PPX1 and PPX2, encode plastid and mitochondrial PPO isozymes, respectively. When susceptible plants are treated with PPO inhibitors, the substrate of PPO, protogen IX, accumulates and is exported from the organelles into the cytoplasm where herbicide-insensitive peroxidase-like enzymes in the plasma membrane convert it to proto IX. Proto IX accumulates in the cytoplasm and, in the presence of light, induces the formation of singlet oxygen that is damaging to cell membranes.
Herbicides that act by inhibiting protoporphyrinogen oxidase are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites (Lermontova et. al., 1997; Watanabe et. al., 2001). Evaluation of a PPO inhibitor-resistant A. tuberculatus biotype revealed that resistance was an incompletely dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid- and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare (Patzoldt et. al., 2006; Gressel and Levy 2006; Tranel et al., 2007).
Even if fluridone/flurochloridone and protox-inhibiting herbicides are known, their use in algal or cyanobacterial culture has not been possible because the cultured photosynthetic algae or cyanobacteria would also be killed. Moreover, there is an unsolved problem of contamination of alga culture ponds and bioreactors with unwanted species such as rotifers and other zooplankton, which are not controlled by phytoene desaturase or protox-inhibiting algae. This disclosure provides solution to each of these unsolved contamination problems.